Tissues freeze-dried sections

C. These freeze-dried sections were dry mounted on microscope slides which had been precoated with either Kodak NTB-3 or NTB-10 emulsion. Other techniques which thawed the frozen section, embedded the tissue in paraffin or dipped the section in liquid emulsion were demonstrated to translocate diffusible compounds. Many other similar attempts have been and are currently being made to localize diffusible compounds by autoradiography at the electron microscope level. 

The detection of serotonin in nervous and non-nervous tissue was aided by the development of the Falck-Hillarp histochemical technique, a method whereby freeze-dried sections of tissue, when exposed to formaldehyde vapour cause indoleamines to emit a yellow fluorescence. Dahlstrom and Fuxe used this technique to show that the highest concentration of serotonin in the brain is located in the raphe nuclei, projections from these cell bodies ascending to the forebrain via the medial forebrain bundle. Descending fibres were also shown to project to the dorsal and lateral horns and the intermediolateral column of the spinal cord. Detailed observation of the distribution of the serotonergic system in the brain became possible with... 

The previously described methods necessitate the extraction of the catecholamines from the tissue prior to assay. The histochemical fluorescence technique allows the visualization of the amines in situ, but it is not an accurate quantitative procedure. Freeze-dried sections of tissue are exposed to formaldehyde vapour at SO C for 1 hr or more. The catecholamines are thereby converted to hydroxyiso-quinoline derivatives (Fig. 1), which fluoresce strongly under U-V light in the fluorescence microscope (Fig. 1, p. 110). ADR and other secondary amines can be distinguished from NA, DA and DOPA by their slower rate of reaction with formaldehyde. 

Prepare sagittal whole-body cryosections (40 (xm thick) using a cryomicrotome. Transfer the frozen sections to glass slides using a tape-transfer process, which utilizes a photoac-tivated polymer adhesive. After transfer to the glass slides, freeze-dry the sections within the chamber of the cryomicrotome. Store all tissue sections in a desiccator at room temperature until analysis. 

A classical example of the confusion that can arise through inadequate technique in autoradiography is that of the localisation of oestradiol in the uterus. Several different groups of investigators produced a remarkably diverse collection of differing results [103-108], Thus, the steroid was either localised in the cytoplasm [104] or in the nucleus [108] of the cells of the endometrium [105] or it was found evenly distributed over all the tissues [104]. Furthermore, it was present [103] or absent in the glandular lumina [108]. However, Stumpf and Roth analysed these differences and evolved a procedure which uses freeze-dried unfixed frozen sections, dry mounted on... 

Apart from the fabrication techniques mentioned in the previous sections, other fabrication techniques such as solvent casting, freeze-drying, and electrodeposition, and hot-melt extrusion have been successfully employed for tissue engineering applications. 

Small pieces of tissues are fixed in PLP for 4 h at 4°C on a rotatory shaker, washed overnight at 4°C in 100 mM phosphate buffer, pH 7.4, containing 10% sucrose, followed by several washings 15% sucrose in buffer (overnight at 4°C), 20% sucrose in buffer (4 h at 4°C), 20% sucrose plus 20% glycerol (1 h at 4°C). The pieces are embedded in OCT medium (Miles), in a small cup of heavy aluminum foil, by snap-freezing in ethanol-dry ice bath while avoiding contact between ethanol and OCT. Frozen sections are cut, 6-12 pm thick, mounted on albuminized slides and washed 3 times 10 min with PBS-10% sucrose. 

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